Process of inactivating rabies virus

ABSTRACT

Process for inactivating a pathogenic rabies virus to produce an apathogenic antigen adaptable to use in the manufacture of a killed vaccine which process comprises freeze drying said rabies virus in the presence of a modified gelatin hydrolyzate containing cross linking urea-alkyleneurea groups and having a molecular weight from about 15,000 to 60,000 and then heating the freeze dried material at a temperature from 30*C to 110*C for 15 minutes to 12 weeks until the pathogenicity thereof is completely destroyed.

United States Patent [1 1 Barth et al.

[ Jan. 7, 1975 PROCESS OF INACTIVATING RABIES VIRUS [75] Inventors:Rudolph Barth; Oskar Jaeger, both of Marburg/Lahn, Germany [73]Assignee: Behringwerke Aktiengesellschaft,

Marburg/Lahn, Germany [22] Filed: May 22, 1973 [21] Appl. No.: 362,820

Related U.S. Application Data [63] Continuation-impart of Ser. No.56,224, July 15, 1970, abandoned, which is a continuation of Ser. No.737,865, June 18, 1968, abandoned.

[30] Foreign Application Priority Data June 22, 1967 Germany 1617350[52] U.S. Cl. 195/12, 424/89 [51] Int. Cl. Cl2k 7/00, A6lk 23/00 [58]Field of Search 195/12; 424/89 [56] References Cited UNITED STATESPATENTS 3,322,632 5/1967 Schwick et al 424/89 FOREIGN PATENTS ORAPPLICATIONS 1,588,829 4/1970 France 195/12 Primary Examiner-Sam RosenAttorney, Agent, or Firm-Curtis, Morris & Safford [57] ABSTRACT Processfor inactivating a pathogenic rabies virus to produce an apathogenicantigen adaptable to use in the manufacture of a killed vaccine whichprocess comprises freeze drying said rabies virus in the presence of amodified gelatin hydrolyzate containing cross linking urea-alkyleneureagroups and having a molecular weight from about 15,000 to 60,000 andthen heating the freeze dried material at a temperature from 30C to 110Cfor 15 minutes to 12 weeks until the pathogenicity thereof is completelydestroyed.

3 Claims, No Drawings 1 PROCESS OF INACTIVATING RABIES VIRUS Thisapplication is a continuation-in-part of our application Ser. No. 56,224filed July 15, 1970 which was a continuation of our application Ser. No.737,865 filed June 18, 1968, both now abandoned.

The present invention relates to a process for inactivating a pathogenicrabies virus.

Processes are already known for the inactivation of biologicalsubstances, in particular of infectious microorganisms and/or theirantigens. For this purpose, either chemical or physical methods havebeen hitherto used. These known processes all have in common aninactivation of the biological substances in the liquid state. Thechemical methods have the disadvantage that for combining the biologicalmaterial with the inactivating substance, the former must be opened andmay be contaminated by fungi and bacteria. In most cases, addedchemicals do not permit exact discontinuation of the inactivation andaffect desired properties, for example the antigenicity of virus andbacteria intended to be used for the manufacture of vaccines. Thestorability of such substances is limited for the same reason. However,the storability of biological material is an important factor, forexample during transportation or in tropical countries where highertemperatures are unavoidable and must be taken into account. Therefore,storage in a refrigerator is prescribed for such preparations. Theattempt has also been made, when formaldehyde is used as theinactivating agent, to add bisulfite for binding the unreactedformaldehyde; but this again required opening of the biologicalmaterial. Another disadvantage has also been the fact that the chemicalsadded, for example phenol, cause intolerance reactions in humans andanimals.

The physical methods, too, have disadvantages. Thus, for example, forinactivating suspensions which contain the mentioned biologicalsubstances, these suspensions must be subjected in a uniformly thickfilm to ultraviolet irradiation in a special apparatus. This may cause,under certain circumstances, formation of peroxides which may continuethe inactivation for an undesirably long period of time.

The object of the present invention is a process for inactivating apathogenic rabies virus which process comprises freeze drying saidrabies virus in the presence of a modified gelatin hydrolyzatecontaining cross linking urea-alkylene-urea groups and having amolecular weight from about 15,000 to 60,000 and then heating the freezedried material at a temperature from 30C to 110C for 15 minutes to 12weeks until the pathogenicity thereof is completely destroyed.

A further object of the present invention is the inactivated rabiesvirus prepared according to the above process and serving for themanufacture of vaccines.

The process of the present invention is novel, since no method hashitherto been known which would permit the inactivation of biologicalmaterial such as infectious microorganisms and/or their antigens in dryform by the action of heat. As has already been pointed out above,inactivation according to known methods has always been carried out inthe liquid phase, either with the aid of suitable chemical agents orphysically, for example by means of ultraviolet irradiation.inactivation of biological material by the action of heat has generallybeen considered with a prejudice because earlier tests had shown thatinactivation in the liquid phase generally caused denaturation of thematerial and therewith impairment of desired biological properties, oreven caused destruction of these properties.

Thus, it was surprising that inactivation according to the presentinvention by the action of heat on dry biological material does notcause its denaturation.

Whereas chemical substances, for example phenol, formaldehyde, etc., areused as inactivating agents in the known methods of inactivation, nosuch chemical agents are added in the process of the present invention,so that side effects such as intolerance reactions, which are due to thepresence of the inactivation agents, do not occur when the preparationsare administered medicinally. Furthermore, the method of the presentinvention has the advantage that no risk of bacterial contamination ofthe biological material during the inactivation process is incurred,since the inactivation can be carried out in a closed vessel. ln orderto discontinue the inactivation, it is only necessary to cool thematerial to a lower temperature, for example to room temperature.Another technical advantage is that the preparations prepared accordingto the present invention have a stronger activity than the knownpreparations and that they may be exposed to such temperatures at whichthe vaccines of the state of the art are already inactivated.

For preparing a vaccine from the inactivated rabies virus preparedaccording to the present invention, the dry preparation is simplycombined with distilled water, up to the desired concentration, ifdesired with the addition of a suitable buffer, for example a phosphateso lution which may also contain an adjuvant, for example aluminumhydroxide. The vaccine thus obtained permits the production in knownmanner of immunities against rabies.

Furthermore, the vaccines prepared according to the present inventionhave the advantage over the vaccines obtained by known methods that theyare considerably more stable. While the vaccines prepared according toknown methods because of their sensitivity to higher temperatures haveto be stored and transported at temperatures in proximity of thefreezing point (0 4C), which, for example under tropical conditions, isconnected with difficulties, the vaccines prepared according to theprocess of the present invention are well storable also at elevatedtemperatures, for example at C, even over prolonged periods of time.

The process of the present invention is suitably carried out bycombining a conventionally prepared aqueous suspension of rabies virushaving antigenic action, with a stabilizing substance as defined below,then freeze-drying it and heating the dry preparation obtained for aperiod from 15 minutes up to 12 weeks, preferably for a period from 60minutes to 10 weeks, to a temperature from 30 to l 10C, preferably to35100C.

As a stabilizer, a polymerisation product prepared according to GermanPat. No. 1,1 18,792 from degraded gelatin, which is commerciallyavailable under the Registered Trade mark Haemaccel is used.

The duration of the action of heat is in inverse proportion to thetemperature at which the inactivation according to the invention iscarried out, i.e., the higher the inactivation temperature, the shorterthe time required for complete inactivation. For example, theinactivation of rabies viruses in the presence of Haemaccel as thestabilizer at 37C takes 60 days, whereas at 56C it requires only 7 days.

After treatment according to the present invention, the preparations aresubjected to the usual tests in order to determine whether thepathogenicity is removed and whether the antigenicity is maintained.

Thus, innocuousness is tested by intracerebral injec-- EXAMPLE:

20 g of wet substance recovered from the brain and the spinal cord of arabbit infected with fixed rabies virus were combined with 180 ml of a3.5 percent solution of a polymeric gelatin derivative, commerciallyavailable under the Registered Trade mark Haemaccel. The mixture wasblended in a homogenizer to a fine suspension, filled into flasks havinga capacity of 7 ml and freeze-dried.

The virus concentration of the dried material, which had beenreconstituted to 7 ml was checked in mice having a weight in the rangeof 11 15 g by intracerebral (i.e.) titration. It was found to be IO LDper 0.03 ml.

For inactivation of the virus material, the flasks were then heated for7 days to 56C; Macroscopically, the action of heat did not change thedry material. After 7 days the material was diluted with distilled waterto the original concentration (7 ml) and the innocuousness and theactivity were tested in mice having a weight ranging from 11 15 g in thefollowing manner:

Test for innocuousness in a. Mice:

Twenty Mice received 0.03 ml of the dissolved material by intracerebralinjection. Within an observation period of 14 days, the animals showedno signs of rabies. The virus material was thus inactivated. The resultwas confirmed by several tests.

b. Dogs:

Two Dogs received subcutaneously, on 6 consecutive days, 4 ml of thevaccine prepared according to the present invention. During anobservation period of 21 days, neither local nor general reactions wereobserved.

c. Guinea pigs:

Five Guinea pigs were administered intraperitoneally 2 ml each of thevaccine prepared according to the present invention. During anobservation period of days, neither local nor general reactions wereobserved.

Six Guinea pigs were administered subcutaneously 0.5 ml each of thevaccine to be tested. During an observation period of 10 days, no localreactions were observed.

d. Rabbits:

Five rabbits were administered intracerebrally 0.3 ml each of thevaccine prepared according to the present invention.

During the observation period of 42 days, no reactions were observed.The result was confirmed by repeated tests carried out under the sameconditions and with the same number of animals.

The above-described innocuousness tests showed that the rabies vaccineprepared according to the present invention is innocuous and welltolerated. Activity:

a. Mouse The activity of the rabies vaccine was determined according to'the so-called Habel test- To carry out this test, 60 mice were immunizedby 6 intraperitoneal (i.p.) injections of 0.25 ml each of a suspension,which was obtained by diluting to a ratio of l 20 the content of a flask(7 ml) with phosphate-buffered distilled water or physiological NaClsolution, and which thus contained the brain and spinal cord material ina concentration of 0.5 percent. The injections were given each time onMonday, Wednesday and Friday of two consecutive weeks. 15 Days after thefirst vaccination, the mice were subjected to a challenge infection. Forthis purpose, 10 mice each were infected, intracerebrally (i.c.), with0.03 ml of the virulent rabies test virus CVS 27 I b 2 (Challenge virusstrain) in dilutions from l0 up to 10*. At the same time, 10 untreatedmice were infected with dilutions from 10 to 10 The 50 percent endpoints of both test series were then determined according to Reed andMiinch [cf. Am. J. Hyg. 27, 493 (1938)]. The difference between bothfinal points is the PD i.e. the dose which is capable of protecting 50percent of the animals against an infection of the indicated magnitude.A vaccine is considered as having passed the Habel test if thedifference between both 50 percent end points of'the vaccinated groupand of the control group, i.e., the PD reaches a value higher than 10 ormore. The vaccine prepared according to the present invention gave insev eral tests a titer of 10- to 1O- PD per 0.03 ml. b. Dog

The vaccine prepared according to the present invention (A 12) wasadministered subcutaneously, in doses of l X 10 ml and l X 5 ml, togroups of 2 dogs each, the sera of which, at the beginning of the test,were free from virus-neutralizing antibodies against rabies. The courseof the immunity was controlled over a period of 52 weeks by examinationof the mixed sere of the groups of dogs by periodical withdrawal ofblood and determination of the antibodies by serum neutralization testsin a white mouse. 14 Days after the immunization, the maximum ofimmunity was already reached with titers between 1 l2 and l (50 percentneutralizing end point of the serum dilution, i.e. this dilutionneutralizes 50 percent of a same volume of a virus suspension of theinfectious rabies virus) and then receded slowly over a period of 50weeks. At the end of the test, 52 weeks after the immunization, the seraof the groups of dogs had the following values:

Dog No. Vaccination Serum titer (50 end point) 52 weeks aftervaccination 6053 l X 10 ml l:l2.3 6083 6190 lX5ml 1:72 6191 weredetermined, at the end of the test (52 weeks after the vaccination) aserum titer (50 percent end point) of-l 3. This test, too, showed thatthe vaccine prepared according to the present invention had a betteractivity than the known vaccines. Stability The vaccine preparedaccording to the present Example was stored for 60 days at 56C andcompared with a rabies vaccine prepared by inactivation with phenol andstored at the lower temperature of 37C.

The vaccine of the present invention exhibited no loss of activity after60 days at 56C whereas the vaccine prepared in known manner after daysat the lower temperature of 37C already showed an activity of only 10"PD 50 per 0.03 ml.

As is evident from the above, the vaccine prepared according to thepresent invention withstands higher temperatures considerably betterwhereas the vaccine Temperature Duration of inactivation 37C 60 days 45Cdays 56C 7 days EXAMPLE 4 Rabies virus were combined with Haemacell andinactivated by heating to 98C, as indicated in Examples 1 and 2. Thefollowing Table shows the test results.

Composition of the rabies Method of inactivation Test for inactivityResult of the vaccine Temperature Time in a mouse activity test i.c.injection in Habel test 20 g of a rabbits brain 40 ml of physiol.NaCl-solution ml of sacch.-glutamate- 98C min. no rabies virus 4.3

solution 90 ml of Haemaccel found used for comparison had already lostits antigenicity at 30 a lower temperature.

EXAMPLE 2 A vaccine prepared as described in Example 1 from rabiesviruses, which had been stabilized with Haemaccel, was subjected to theHabel test as well as to the stability test. The test results withindication of the test conditions are shown in the following Table. Forcomparison, two vaccines inactivated in known manner 40 with phenol wereused.

It has been shown in this Example that even extreme temperatures may beused for inactivation without impairing the activity of the vaccine ofthe present invention. The inactivation time is thereby essentiallyreduced.

EXAMPLE 5 Three vaccines, prepared in accordance with the presentinvention, i.e.

A 16 (inactivated for 5 days at +70C) A 17 (inactivated for 3 days at+70C) A 18 (inactivated for 7 days at +70C),

The test results show that the vaccine containing rabies virusesinactivated according to the process of the present invention resist aconsiderably higher temperature than the vaccine prepared according toknown processes.

EXAMPLE 3 Inactivation tests were carried out at various temperatureswith samples of the material indicated in Example 1 and the followingresults were obtained:

were tested and compared in the Habel test with two rabies vaccinesprepared by inactivation with phenol according to Hemp? (B and 8,).

As has already been explained in Example 1, the Habel test requires that6 injections of 0.25 ml each of the vaccine, diluted to 0.5 percent ofthe nerve tissue proportion, protects against 1,000 LD (log 3) of theinfectious virus. With the rabies vaccine according to Hempt, therefore,the 10 percent nerve tissue suspension is normally diluted in the ratioof 1 z 20..

Vaccine Preparation Vaccine dilution of nerve tissue proportion l:20(0.51:40 (0.25 1:80 (0.125

A 16 according to a 5.7 5.7 5.7 the invention b 1.0 1.4 2.0 c 4.7 4.33.7 A 17 according to a 5.7 5.7 5.7 the invention b 1.4 2.4 2.1 c 4.33.3 3.6 A 18 according to a 6.1 6.1 6.1 the invention b 1.0 1.0 1.6 c5.1 5.1 4.5 B 3 according to a 5.3 5.3 5.3 state of the b 1.6 2.0 3.3art c 3.7 3.3 2.0 B 4 according to a 5.7 5.7 not state of the b 2.6 3.6tested art c 3.1 2.1

a =1og. of the 50 end point of the control animals (LDW) b log. of the50 I: end point of the vaccinatedanimals (L050) 0 log. of the PD (3 b)It can be seen from the above Table that the vaccines prepared accordingto the present invention in the dilutions 1 20, 1 40 and even 1 80comply with the requirements of the Habel test. The rabies vaccinesprepared according to l-lempt corresponding to the state of the art, ina dilution of l 20, comply with the requirements of the Habel test andin the dilution of l 40, only one vaccine passed the test, and in thedilution of 1 80 this vaccine no longer complied with the requirementsof the Habel test. Thus, the vaccines of the present invention have afar better antigenicity than the vaccines prepared according to thestate of the art.

H 1 490 99! iqt esti atipa p heesnis rabies v r which process comprisesfreeze drying said rabies virus in the presence of a gelatin hydrolyzatecontaining cross linking urea-alkylene-urea groups and having amolecular weight from about 15,000 to 60,000 and then heating the freezedried material at a temperature from 30C to 110C for 15 minutes to 12weeks until the pathogenicity thereof is completely destroyed.

2. A method as in claim 1 wherein said freeze-dried material is heatedfor one hour to ten weeks until the pathogenicity thereof is completelydestroyed.

3. A method as in claim 1 wherein said freeze-dried material is heatedto a temperature from 35C. to C until the pathogenicity thereof iscompletely destroyed.

1. A METHOD FOR INACTIVATING A PATHOGENIC RABIES VIRUS WHICH PROCESSCOMPRISES FREEZE DRYING SAID RABIES VIRUS IN THE PRESENCE OF GELATINHYDROLYZATE CONTANING CROSS LINKING UREAALKYLENE-UREA GROUPS AND HAVINGMOLECULAR WEIGHT FROM ABOUT 15,000 TO 60,000 AND THEN HEATING THE FREEZEDRIED MATERIAL AT A TEMPERATURE FROM 30*C TO 110*C FOR 15 MINUTES TO 12WEEKS UNTIL THE PATHOGENICITY THEREFORE IS COMPLETELY DESTROYED.
 2. Amethod as in claim 1 wherein said freeze-dried material is heated forone hour to ten weeks until the pathogenicity thereof is completelydestroyed.
 3. A method as in claim 1 wherein said freeze-dried materialis heated to a temperature from 35*C. to 100*C until the pathogenicitythereof is completely destroyed.